The proposed research is aimed at obtaining insights into the mechanisms by which two bone-specific proteins, bone morphogenetic protein and bone inhibitory protein, regulate the differentiation of mesenchymal cells along the pathway culminating in new bone formation and whether these proteins are involved in the maintenance of the differentiated state. These two proteins have been partially purified and both exhibit biological activity in vivo and in vitro. Our specific intent is to purify to homogeneity these two proteins and to study their effects on bone formation and inhibition of this process and whether or not they influence the differentiation of mesenchymal cells in vitro. We will also attempt to define which cell is responsible for the synthesis of these proteins by testing for their production by osteosarcoma cells, which display osteoblastic characteristics in vitro. To this end, the proteins will be purified, antibodies will be produced against these proteins, and these will be used as probes to define whether osteosarcoma cells produce the proteins. In addition, using cultures of undifferentiated chick limb bud mesenchyme and various molar ratios of these two proteins, bone morphogenetic protein and bone inhibitory protein, expression of chondrogenic and osteogenic phenotype will be examined. The biochemical markers to be used as evidence of chondrogenesis are glycosaminoglycan and type II collagen synthesis; for osteogenesis, alkaline phosphatase activity and incorporation of 45Ca into mineral. The information obtained from such studies is important for elucidating factors which may influence differentiation of bone, but more importantly, such information will be useful in understanding basic biological mechanisms involved in fracture healing. Such information, by extrapolation, will yield insight into aberrant regulation of bone formation such as observed in ectopic bone formation as a result of injury, insufficient bone formation observed in degenerative changes associated with aging, as well as age-related pathological changes seen in diseases such as peridontal disease and osteoporosis.